1. Biocompatible Polymers
Biocompatible polymeric materials have been used extensively in therapeutic drug delivery and medical implant device applications. In certain applications, it may be desirable for such polymers to be not only biocompatible, but also biodegradable.
A biocompatible, biodegradable polymer is one effective means of delivering a therapeutic agent or other biologically active substance. See generally Langer et al., (1983) Rev. Macro. Chem. Phys. C23(1):61. Polymers have been used as carriers of therapeutic agents to effect a localized and sustained release. See generally Controlled Drug Delivery, Vols. I and II; Bruck et al., eds. (1982); and Chien et al., (1982) Novel Drug Delivery Systems. Such delivery systems may provide enhanced therapeutic efficacy and reduced overall toxicity.
Typically, a biodegradable matrix not only allows for diffusional release of any therapeutic or other agent, but also allows for controlled release of such agent by degradation of the polymer matrix. Specific examples of biodegradable materials that have been used, or proposed for such use, include: polylactide, polyglycolide, polydioxanone, poly(lactide-co-glycolide), poly(glycolide-co-polydioxarone), polyanhydride, poly(glycolide-co-trimethylene carbonate), and poly(glycolide-co-caprolactone).
Polymers having phosphorous linkages are known. Some respective structures of such polymers, each of which have a different sidechain connected to the phosphorus atom, are: 
In part, the present invention provides biocompatible polymers having phosphorous-based linkages and related compositions and formulations, and methods for using them.
2. Gene Therapy
Somatic gene therapy may be defined, in part, as the ability to cause ectopic expression of a gene in non-germ line cells of an animal or patient by the introduction of foreign nucleic acid. In general, gene therapy may be divided into two categories. Ex vivo gene therapy typically involves the removal of cells from a host organism, introduction of a foreign gene or other nucleic acid into those cells in the laboratory, and reimplantation or transplantation of the modified cells back into a recipient host. In contrast, in vivo gene therapy generally involves the introduction of a foreign gene or other nucleic acid directly into cells of a recipient host without the need for prior removal of those cells.
A. Gene Therapy in Cardiac Myocytes
The ability to program gene expression in cardiac myocytes may enable the treatment of a number of inherited, acquired or other cardiac diseases. Applications of this approach may be divided into several general categories, two of which are described in more detail below
As many as 1.5 million patients per year in the U.S. suffer a myocardial infarction (MI). Many millions more suffer from syndromes of chronic myocardial ischemia due to large and small vessel coronary atherosclerosis. Many of these patients may benefit from the ability to stimulate collateral vessel formation in areas of ischemic myocardium. Such a result may be achieved, for example, by injecting plasmids encoding recombinant angiogenesis factors directly into the left ventricular wall to stimulate new collateral circulation in areas of chronically ischemic myocardium. Also, this approach maybe used to study directly the molecular mechanisms regulating cardiac myocyte gene expression both during cardiac myogeneses and in a variety of pathophysiologic states such as cardiac hypertrophy. Gene therapy methods may provide an alternative approach to the current methods of coronary artery bypass and percutaneous transluminal coronary angioplasty. For certain patients having severe and diffuse atherosclerosis that they are not candidates for bypass or angioplasty.
A number of genetic disorders affect myocardial performance. For example, many patients with Duchenne""s muscular dystrophy also suffer from a cardiomyopathy. In addition, it is clear that there are a number of other genetically-inherited cardiomyopathies of unknown etiology. Gene therapy approaches may be useful in treating a variety of these inherited disorders of cardiac function. For example, injection of vectors containing the normal dystrophin gene or cDNA, or their functional equivalent, may correct the defect in patients with Duchenne""s muscular dystrophy. In part, the present invention provides gene therapy compositions for treatment of disorders affecting myocardial and other muscle tissues.
B. Gene Therapy Using Skeletal Myoblasts
A variety of acquired and inherited diseases are currently treated by intravenous or subcutaneous infusions of recombinant or purified proteins as needed. Some examples include diabetes mellitus, which may be treated with subcutaneous or intravenous injections of insulin, hemophilia A, which may be treated with intravenous infusions of factor VIII, and pituitary dwarfism, which may be treated with subcutaneous injections of growth hormone. The development of expression systems that may produce and deliver such recombinant proteins into the systemic circulation represents an important advance in treatment of such diseases.
In certain instances, the recombinant protein delivery system may utilize muscle cells isolated from the recipient, which are grown and transduced with one or more recombinant genes in vitro, and reimplanted into the host organism. Such cells may be engineered to produce large amounts of secreted recombinant protein, which, following secretion, may be circulated.
In part, the present invention provides formulations of gene expression constructs for transfecting skeletal muscle stem cells (myoblasts) to produce therapeutic levels of serum proteins in a recipient host. In one embodiment, for example, myocytes are engineered to secrete recombinant proteins in the myocardium.
3. Genetic Immunization
One means of evoking an immune response involves DNA-based immunization. In one aspect, such immunization involves introducing plasmid DNA, containing protein coding sequences and the necessary regulatory elements to express them, into tissues of the organism by means of a parenteral injection of a simple saline solution. Such application of plasmid DNA to muscular tissues has been shown to lead to transgene expression. DNA immunization may elicit both antibody and cell-mediated immune responses, thereby generating protective immunity.
One aspect typically inherent to this means of immunization is that the immunizing molecule, usually plasmid DNA, is often not highly immunogenic. It appears that some features of DNA-based immunization do mimic, at least in part, the immune response induced by a viral infection. For example antigen presentation by molecules of the major histocompatibility complex (MHC) class I pathway may take place, thus leading to the induction of cytotoxic T lymphocytes.
In part, the present invention presents improved DNA vaccinations, and methods of using them.
Design of controlled release systems and tissue engineering scaffolds continues to stimulate development of new biodegradable polymers. Polymers having phosphate linkages in the backbone have shown promise because of their structural versatility and attractive physico-chemical properties. The present invention is directed in part to the discovery of a polymer system for nucleic acid transfer into muscle and other tissue both in vitro and in vivo. In certain instances, the subject formulations may be used to effectuate both stable and transient transfection of cells. The subject formulations may provide prolonged transgene expression even under transient conditions and in non-dividing cells. In addition, the subject formulations are useful as DNA vaccinations.
In one aspect, the present invention is directed to a polymer formulation or system, methods for treatment using such system, and precursors of the polymer system, such as a liquid composition, all for delivery of nucleic acid and other materials.
In another aspect, the present invention relates to compositions and methods for delivery of nucleic acids in vivo. In one aspect, the present invention provides a polymeric delivery formulation including a nucleic acid to be transfected, formulated in a biodegradable polymer having phosphorous-based linkages. In certain embodiments, such linkages are comprised of poly(phosphoester)s or derivatives or analogs thereof. In other embodiments of the present invention, the polymer matrix, loaded with one or more substances such as nucleic acid or therapeutic agent, may be prepared as a microsphere for use.
The versatility of the polymers of the present invention results in part from the versatility of the phosphorus atom, which is known for a multiplicity of reactions. The physico-chemical properties of the phosphorous linkage may be readily manipulated, in part, by varying substituents attached to the phosphorous atom. In certain embodiments, the degradation observed for any polymer in vivo is due in large part to the physiologically labile phosphoester bond (or other analogs or derivatives thereof) in the backbone of the polymer. By manipulating the backbone or other substituents, a wide range of degradation rates may be attained.
The gene delivery system of the present invention, and methods of using such system, have a variety of attractive features, some of which include the following: (i) ligands may be conjugated to the DNA-nanosphere for potential tissue targeting and to stimulate receptor-mediated endocytosis; (ii) lysosomolytic agents may be incorporated to promote escape of DNA from endosomal and lysosomal compartments; (iii) bioavailability of the DNA may be improved because of protection from serum nuclease degradation by the polymer matrix; (iv) other bioactive agents such as proteins, multiple plasmids, or drugs may be co-encapsulated in the formulations; and (v) prolonged gene expression may allow for a single dose DNA vaccine.
This polymeric system may be administered as is necessary depending on the subject being treated, the severity of the affliction, the judgment of the prescribing physician, and the like.